Engineered Type IV Pilin of Clostridium difficile

ABSTRACT

The present invention relates to engineered  Clostridium difficile  type IV pilin (tfp) genes, type IV pilin proteins which can serve as a diagnostic marker for identification of patients infected with  C. difficile,  and vaccines comprising type IV pilin proteins, antigenic fragments and variants thereof for therapeutic interventions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Appl. No. 61/179,747, filedMay 20, 2009. The content of the aforesaid application is relied uponand incorporated by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. A137606awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention generally relates at least to the fields ofmolecular biology, immunology, infectious disease and medicine. Inparticular, the invention relates to a Clostridium difficile type IVpilin (tfp) gene which can serve as a diagnostic marker foridentification of patients infected with C. difficile and as a vaccinefor therapeutic interventions.

BACKGROUND OF THE INVENTION

Clostridium difficile associated disease (CDAD) is a dangerous emerginginfection caused by an anaerobic, spore-forming bacillus that oftenthreatens the health of elderly patients in various healthcare settingfollowing antibiotic treatments of unrelated infections. CDAD symptomsrange in severity from asymptomatic carriage, through mild diarrhea, toa more severe pseudomembranous colitis which can be fatal. Theepidemiology of CDAD has been changing dramatically. Formerly foundalmost exclusively as a complication of antimicrobial therapy among theelderly and infirm in inpatient settings, CDAD has been reportedincreasingly in outpatients, among otherwise healthy individualsincluding children, and even in the absence of antimicrobial therapy.Deaths attributable to CDAD have quadrupled in the United States from5.7 per million persons in 1999 to 23.7 per million in 2004. Redelings MD, Sorvillo F, Mascola L. Increase in Clostridium difficile—relatedmortality rates, United States, 1999-2004. Emerg Infect Dis. 2007;13:1417-9. Estimates of the cost for treatment for CDAD in the UnitedStates have been dramatically revised upward from $1 billion in 2002 to$3.2 billion in 2007 due to a dramatic increase in the number of casesand increasing severity of the disease.

The mechanisms by which C. difficile colonizes the human colon are notestablished. A surface protein layer has been described which appears toplay a role in binding to tissue culture cells in vitro, but its role invivo has not been established. Calabi, E., Calabi, F., Phillips, A. D. &Fairweather, N. Binding of Clostridium difficile surface layer proteinsto gastrointestinal tissues. Infect Immun (2002):70:5770-5778.Enterotoxins A (TcdA) and B (TcdB) are the primary virulence factors ofC. difficile. They are exoenzymes that monoglucosylate small Rho-likeGTPases, ultimately leading to the disruption of the actin cytoskeletonof colonic intestinal epithelial cells, destruction of tight junction,and apoptosis. Release of cytokines from intoxicated target cells alsoleads to massive infiltration of neutrophils into damaged tissueregions, a hallmark of pseudomembranous colitis.

Although primary CDAD can usually be successfully treated withmetronidazole or vancomycin, metronidazole resistance and refractoryinfections are becoming increasingly common. Furthermore, many patientssuffer recurrent episodes of CDAD, which can be extremely distressingand difficult to manage. An effective vaccine against CDAD is urgentlyneeded for primary and secondary (relapse) prevention. No licensedvaccine is currently available for CDAD. The approach to vaccinationthat has advanced furthest into clinical trials has targeted only the C.difficile enterotoxins. Toxoid preparations of toxin A and B havecompleted phase I trials, with both serum free and fecal antibodyagainst both toxins being demonstrated. Kotloff K L, Wasserman S S,Losonsky G A, Thomas W, Jr., Nichols R, Edelman R, Bridwell M, Monath TP. Safety and immunogenicity of increasing doses of a Clostridiumdifficile toxoid vaccine administered to healthy adults. Infect. Immun.2001 February; 69(2):988-95. The mechanisms by which serum antibodyresponses are effective against infection and disease caused by C.difficile are unclear, although it has been proposed that entry of IgGantitoxin from the blood into mucosal tissues of the large bowel orintestinal lumen may prevent toxin binding.

Type IV pili (Tfp) or fimbriae are hair-like surface appendages producedby many species of Gram negative bacteria including Pseudomonasaeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, N. meningitidis,Salmonella enterica serovar Typhi (herein designated S. typhi),Legionella pneumophila, enteropathogenic and enterotoxigenic Escherichiacoli. Tfp play numerous roles in diverse processes such as cellularadhesion, colonization, twitching motility, biofilm formation, andvirulence. Tfp are composed exclusively of primarily of many copies ofpilin protein, tightly packed in a helix so that the highly hydrophobicamino-terminus of the pilin is buried in the core of the pilus. Tfppilins have been used successfully as subunit vaccines for theprevention of several diseases in animals. Lepper A W, Moore L J, AtwellJ L, Tennent J M. The protective efficacy of pili from different strainsof Moraxella bovis within the same serogroup against infectious bovinekeratoconjunctivitis. Vet. Microbiol. 1992; 32:177-87, Lepper A W D,Atwell J L, Lehrbach P R, Schwartzkoff C L, Egerton J R, Tennent J M.The protective efficacy of cloned Moraxella bovis pili in monovalent andmultivalent vaccine formulations against experimentally inducedinfectious bovine keratoconjunctivitis (IBK). Vet. Microbiol. 1995;45:129-38. Stewart D J, Clark B L, Peterson J E, Emery D L, Smith E F,Griffiths D A, O'Donnell I J. The protection given by pilus and wholecell vaccines of Bacteroides nodosus strain 198 against ovine foot-rotinduced by strains of different serogroups. Aust. Vet. J. 1985;62:153-9. Egerton J R, Cox P T, Anderson B J, Kristo C, Norman M,Mattick J S. Protection of sheep against footrot with a recombinantDNA-based fimbrial vaccine. Vet. Microbiol. 1987; 14:393-409. Recentlyinvestigators have discovered that Clostridium perfringens has the genesfor and can produce Tfp; similar genes are present in the C difficilegenome. Varga J J, Nguyen V, O'Brien D K, Rodgers K, Walker R A,Melville S B. Type IV pili-dependent gliding motility in theGram-positive pathogen Clostridium perfringens and other Clostridia.Mol. Microbiol. 2006 November; 62(3):680-94.

There is a need for a multivalent C. difficile subunit vaccine and adiagnostic marker for identification of patients infected with C.difficile.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a method of inducingan immune response, comprising administering to a subject in needthereof an immunologically-effective amount of a vaccine comprising aClostridium difficile type IV pilin or an antigenic fragment or variantthereof.

In another embodiment, the present invention relates to a method foroverexpression of multiple C. difficile type IV pilins in gram negativebacteria including but not limited to E. coli. The type IV pili serve ascolonization factors and vaccine targets.

In another embodiment, the present invention relates to a method forexpressing and purifying high levels of C. difficile type IV pilins.

In another embodiment, the present invention relates to novel Type IVpilin proteins of C. difficile which exhibit high level expression inthe bacteria E. coli.

In another embodiment, the present invention relates to novel vaccinesfor C. difficile comprising engineered recombinant C. difficile type IVpilins.

In another embodiment, the present invention relates to novel biomarkersfor use in C. difficile detection in patients with C. difficileinfections.

In another embodiment, the present invention relates to a method forprevention of C. difficile colonization and disease in a subjectcomprising administering a vaccine comprised of purified type IV pilinproteins to said subject.

In another embodiment, the present invention relates to a method forprevention of C. difficile spread among mammalian hosts, such as humans,comprising administering a vaccine comprised of purified type IV pilinproteins to said mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Strategy for modification and purification of C. difficile PilA2type IV pilin. The nucleotide and corresponding amino acid sequences ofthe native and recombinant pilA2 genes are aligned. The hexahistidinetag, enterokinase cleavage sequences of the recombinant protein,pre-pilin peptidase signal sequence and hydrophobic mature aminoterminus of the native protein are boxed.

FIG. 2. Coomassie stained gel of purified C. difficile PilA1 and PilA2proteins. M, molecular size standards are shown in the first lane.

FIG. 3. PilA1 is immunogenic in mice. Geometric mean titers for fivemice immunized as described in the text. Data are geometric means andstandard errors of triplicate samples from three independent ELISAassays.

DETAILED DESCRIPTION OF THE INVENTION

Multiple Pilin Genes and Alleles Identified in Various Strains of C.difficile.

In one aspect, the present invention is directed to an engineered TypeIV pilin gene of C. diffcile.

In accordance with the claimed invention, the type IV pilin can comefrom any C. difficile strain. Examples of C. difficile strains includeC. difficile CD196, C. difficile CIP 107932, C. difficile QCD-32g58, C.difficile QCD-37x79, C. difficile QCD-66c26, C. difficile QCD-76w55, C.difficile QCD-97b34, C. difficile R20291, C. difficile QCD-63q42, C.difficile QCD-23m63, C. difficile 630, and C. difficile ATCC 43255.

All of the strains listed above have two to four genes that can bepredicted to encode type IV pilin proteins. As used herein, these geneswill be referred to as pilA1, pilA2, pilA3 and pilA4. The C. difficilestrains can harbor allelic variants of the type IV pilins. Alleles ofpilA1, as used herein, include pilA1.1, pilA1.2, pilA1.3, pilA1.4 andpilA1.5. Alleles of pilA2, as used herein, include pilA2.1 and pilA2.2.Alleles of pilA3, as used herein, include pilA3.1, pilA3.2 and pilA3.3.Alleles of pilA4, as used herein, include pilA4.1.

Each of these genes is predicted to encode a protein composed of ashort, positively charged signal peptide, a prepilin peptidase cleavagesite and a hydrophobic mature amino-terminal domain characteristic ofType IVa pilins. Mature pilin proteins are naturally derived frompre-pilin proteins by the action of pre-pilin peptidase enzymes whichcleave the signal peptide and N-methylate the mature amino terminus.

PilA1: The mature PilA1 protein is predicted to be 158-164 amino acidslong. Eight strains are predicted to express identical PilA1 proteins,while the PilA1 proteins of the other four strains are predicted to be75%, 89%, 91% and 93% identical to these eight.

PilA1.1 is found in C. difficile strains CD196, CIP 107932, QCD-32g58,QCD-37x79, QCD-66c26, QCD-76w55, QCD-97b34 and R20291. The nativenucleotide sequence of PilA1.1 is SEQ ID NO:1 and the native amino acidsequence is SEQ ID NO:2.

PilA1.2 is found in C. difficile strain QCD-63q42. The native nucleotidesequence of PilA1.2 is SEQ ID NO:5 and the native amino acid sequence isSEQ ID NO:6.

PilA1.3 is found in C. difficile strain QCD-23m63. The native nucleotidesequence of PilA1.3 is SEQ ID NO:9 and the native amino acid sequence isSEQ ID NO:10.

PilA1.4 is found in C. difficile strain 630. The native nucleotidesequence of PilA1.4 is SEQ ID NO:13 and the native amino acid sequenceis SEQ ID NO:14.

PilA1.5 is found in C. difficile strain ATCC 43255. The nativenucleotide sequence of PilA1.5 is SEQ ID NO:17 and the native amino acidsequence is SEQ ID NO:18.

The first 9 amino acids of SEQ ID NOS: 2, 6, 10, 14, and 18 comprisepre-pilin leader sequence that is cleaved during processing in C.difficile.

PilA2: All 12 strains are predicted to encode a 109-amino acid maturePilA2 protein. The predicted PilA2 protein is identical in 11 of thesestrains, and 95% identical in the other strain.

PilA2.1 is found in C. difficile strains CD196, CIP 107932, QCD-32g58,QCD-37x79, QCD-66c26, QCD-76w55, QCD-97b34, R20291, QCD63q42, 630 andATCC 43255. The native nucleotide sequence of PilA2.1 is SEQ ID NO:21and the native amino acid sequence is SEQ ID NO:22. The first 11 aminoacids of SEQ ID NO:22 comprise pre-pilin leader sequence that is cleavedduring processing to yield the mature protein.

PilA2.2 is found in C. difficile strain QCD-23m63. The native nucleotidesequence of PilA2.2 is SEQ ID NO:25 and the native amino acid sequenceis SEQ ID NO:26. The first 8 amino acids of SEQ ID NO:26 comprisepre-pilin leader sequence that is cleaved during processing to yield themature protein.

PilA3: The pilA3 gene is present in 11 of the 12 strains and ispredicted to encode a protein closely related at its amino terminus toPilA1. Forty-two of the first 57 amino acids of all PilA1 and PilA3predicted mature proteins are identical. However, the sequencesimilarities between PilA1 and PilA3 end abruptly at that point. Thepredicted mature PilA3 proteins are 156-159 amino acids long. Sevenstrains are predicted to express identical PilA3 proteins, twoadditional strains are predicted to encode identical PilA3 proteins thatare 76% identical to these seven and one strain is predicted to encode aPilA3 protein 95% identical to those of the majority.

PilA3.1 is found in C. difficile strains CD196, CIP 107932, QCD-32g58,QCD-37x79, QCD-66c26, QCD-76w55, QCD-97b34 and R20291. The nativenucleotide sequence of PilA3.1 is SEQ ID NO:29 and the native amino acidsequence is SEQ ID NO:30.

PilA3.2 is found in C. difficile strain 630. The native nucleotidesequence of PilA3.2 is SEQ ID NO:33 and the native amino acid sequenceis SEQ ID NO:34.

PilA3.3 is found in C. difficile strains QCD-63q42 and ATCC 43255. Thenative nucleotide sequence of PilA3.3 is SEQ ID NO:37 and the nativeamino acid sequence is SEQ ID NO:38.

The first 6 amino acids of SEQ ID NOS:30, 34 and 38 comprise pre-pilinleader sequence that is cleaved during processing to yield the matureproteins.

PilA4: The gene encoding the PilA4 protein is present in only 3 strainsand is predicted to encode an identical 263 amino acid protein. PilA4.1is found in C. difficile strains CD196, QCD32g58 and R20291. The nativenucleotide sequence of PilA4.1 is SEQ ID NO:41 and the native amino acidsequence is SEQ ID NO:42. The first 10 amino acids of SEQ ID NO:42comprise pre-pilin leader sequence that is cleaved during processing toyield the mature protein.

In total, one strain has only two pilA genes, eight strains have threepilA genes and three strains have four.

Vectors, Host Cells, Recombinant Expression, Polypeptides, AntigenicFragments and Variants.

In some embodiments, the present invention relates to vectors thatcomprise a type IV pilin polynucleotide from C. difficile, host cellswhich are genetically engineered to express type IV pilins and theproduction of polypeptides of the invention by recombinant techniques.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the invention.

When a polynucleotide encoding a type IV pilin of C. difficile is usedfor the recombinant production of a polypeptide, the polynucleotide mayinclude the coding sequence for the full-length polypeptide or anantigenic fragment thereof, by itself; the coding sequence for thefull-length polypeptide or fragment in reading frame with other codingsequences, such as those encoding a leader or secretory sequence, apre-, or pro or preproprotein sequence, or other fusion peptideportions. For example, a marker sequence that facilitates purificationof the fused polypeptide can be encoded. In certain embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, for example, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc Natl Acad Sci USA 86:821-824 (1989), orit may be the HA tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson, I., et al., Cell 37:767, 1984).The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, ribosome binding sites andsequences that stabilize mRNA.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces, Bacillussubtilis, and Salmonella enterica typhi cells; fungal cells, such asyeast cells and Aspergillus cells. In some embodiments, gram negativebacteria are the host cells. A great variety of expression systems canbe used, including DNA or RNA vectors.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a type IV pilin operably linked to a heterologouspromoter. In some embodiments, the invention further provides anisolated nucleic acid molecule comprising a type IV pilin operablylinked to a heterologous promoter, wherein said isolated nucleic acidmolecule is capable of expressing a type IV pilin polypeptide when usedto transform an appropriate host cell.

In some embodiments, the invention relates to an isolated nucleic acidmolecule encoding an antigenic fragment of type IV pilin linked to anaffinity tag sequence and enzymatic cleavage sequence to facilitatepurification. In some embodiments, the affinity tag is a 6×-Histidinetag and the cleavage sequence is recognized by enterokinase. In someembodiments, the nucleic acid molecules are optimized to increaseexpression in E. coli without altering the amino acid sequence usingpreferred codons in E. coli.

In some embodiments, the present invention is directed to purifiedpolypeptides, variants and and antigenic fragments of a type IV pilin ofC. difficile. In some embodiments, the type N pilin is engineered tolack the native leader sequence and amino terminal hydrophobic domain.

In some embodiments, the type N pilin polypeptides of the presentinvention include the polypeptides of SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ IDNO:30, SEQ ID NO:34, SEQ ID NO:38 and SEQ ID NO:42 as well as antigenicfragments and variants which have at least 90% identity thereto. In someembodiments, the polypeptides have at least 96%, 97% or 98% identity tothe polypeptides of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ IDNO:34, SEQ ID NO:38, SEQ ID NO:42 and antigenic fragments thereof. Insome embodiments, the polypeptides have at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to the polypeptide of SEQ IDNO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ IDNO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38 and SEQ IDNO:42 and antigenic fragments thereof.

In some embodiments, the variant polypeptides, including those whichhave 90% or more identity to the type IV pilins described herein orantigenic fragments thereof, are recognized by an antibody that binds apolypeptide selected from the group consisting of SEQ ID NO:2, SEQ IDNO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:42 andantigenic fragments thereof. In some embodiments, the invention isdirected to a variant having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to a type IV pilin described herein and isrecognized by an antibody that binds a type IV pilin antigenic fragmentselected from the group consisting of amino acids 35-173 of SEQ NO:2,amino acids 35-173 of SEQ ID NO:6, amino acids 35-173 of SEQ ID NO:10,amino acids 35-171 of SEQ ID NO:14, amino acids 35-170 of SEQ ID NO:18,amino acids 34-119 of SEQ ID NO:22, amino acids 31-116 of SEQ ID NO:26,amino acids 32-164 of SEQ ID NO:30, amino acids 32-164 of SEQ ID NO:34,amino acids 32-162 of SEQ ID NO:38, and amino acids 36-272 of SEQ IDNO:42.

In some embodiments, the type IV pilin polypeptides, variants orantigenic fragments are part of a larger protein such as a fusionprotein. It is often advantageous to include additional amino acidsequence which contains secretory or leader sequences, pro-sequences,sequences which aid in purification such as multiple histidine residues,or additional sequence for stability during recombinant production.

An antigenic fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof one of the aforementioned type IV pilin polypeptides. The antigenicfragment can be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region.

In some embodiments, the antigenic fragments include, for example,truncation polypeptides having the amino acid sequence of the type IVpilin polypeptides, except for deletion of a continuous series ofresidues that includes the amino terminus, or a continuous series ofresidues that includes the carboxyl terminus or deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. In some embodiments, fragments arecharacterized by structural or functional attributes such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, and high antigenic index regions.

The antigenic fragment can be of any size. In some embodiments thefragment is capable of inducing an immune response in a subject or berecognized by a specific antibody. In some embodiments, the fragmentcorresponds to an amino-terminal truncation mutant. In some embodiments,the number of amino terminal amino acids missing from the fragmentranges from 1-100 amino acids. In some embodiments, it ranges from 1-75amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-25amino acids, 1-20 amino acids, 1-15 amino acids, 1-10 amino acids and1-5 amino acids.

In some embodiments, the fragment corresponds to carboxyl-terminaltruncation mutant. In some embodiments, the number of carboxyl terminalamino acids missing from the fragment ranges from 1-100 amino acids. Insome embodiments, it ranges from 1-75 amino acids, 1-50 amino acids,1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids,1-15 amino acids, 1-10 amino acids and 1-5 amino acids.

In some embodiments, the fragment corresponds to an internal fragmentthat lacks both the amino and carboxyl terminal amino acids. In someembodiments, the fragment is 7-200 amino acid residues in length. Insome embodiments, the fragment is 10-100 amino acid residues, 15-85amino acid residues, 25-65 amino acid residues or 30-50 amino acidresidues in length. In some embodiments, the fragment is 7 amino acids,10 amino acids, 12 amino acids, 15 amino acids, 20 amino acids, 25 aminoacids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids,50 amino acids 55 amino acids, 60 amino acids, 80 amino acids or 100amino acids in length.

Of course larger antigenic fragments are also useful according to thepresent invention, as are fragments corresponding to most, if not all,of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30,SEQ ID NO:34, SEQ ID NO:38 and SEQ ID NO:42.

In some embodiments, the antigenic fragment is selected from the groupconsisting of a peptide comprising amino acids 35-173 of SEQ ID NO:2,amino acids 35-173 of SEQ ID NO:6, amino acids 35-173 of SEQ ID NO:10,amino acids 35-171 of SEQ ID NO:14, amino acids 35-170 of SEQ ID NO:18,amino acids 34-119 of SEQ ID NO:22, amino acids 31-116 of SEQ ID NO:26,amino acids 32-164 of SEQ ID NO:30, amino acids 32-164 of SEQ ID NO:34,amino acids 32-162 of SEQ ID NO:38, and amino acids 36-272 of SEQ IDNO:42.

Thus, the polypeptides of the invention include polypeptides having anamino acid sequence at least 90% identical to that of SEQ ID NO:2, SEQID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, SEQ IDNO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38 and SEQ ID NO:42 orantigenic fragments thereof. In some embodiments, the variants are thosethat vary from the reference by conservative amino acid substitutions,i.e., those that substitute a residue with another of likecharacteristics. Typical substitutions are among Ala, Val, Leu and Ile;among Ser and Thr; among the acidic residues Asp and Glu; among Asn andGln; and among the basic residues Lys and Arg, or aromatic residues Pheand Tyr. In some embodiments, the polypeptides are variants in whichseveral, 5 to 10, 1 to 5, or 1 to 2 amino acids are substituted,deleted, or added in any combination.

The type IV pilin polypeptides, variants and antigenic fragments of theinvention can be prepared in any suitable manner. Such polypeptidesinclude isolated naturally occurring polypeptides, recombinantlyproduced polypeptides, synthetically produced polypeptides, orpolypeptides produced by a combination of these methods.

In some embodiments, the invention is directed to engineered type IVpilin which is optimized for high level expression in E. coli usingcodons that are preferred in E. coli. In some embodiments, the inventionis directed to engineered antigenic fragments of type IV pilin of C.difficle (nucleic acid and amino acid sequences), which are optimizedfor expression in E. coli, and harbor a histidine tag and enterokinasecleavage site to facilitate purification of the protein. In someembodiments, the fragments lack the pre-pilin leader sequence andhydrophobic domain found in the native proteins.

In some embodiments, the codons are optimized for high level expressionin E. coli. As used herein, a codon that is “optimized for high levelexpression in E. coli” refers to a codon that is relatively moreabundant in E. coli in comparison with all other codons corresponding tothe same amino acid. In some embodiments, at least 40% of the codons areoptimized for high level expression in E. coli. In some embodiments, atleast 50%, at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% of the codons are optimized forhigh level expression in E. coli.

The following sequences are optimized for expression in E. coli and alsoare fused to histidine tags and enterokinase cleavage sites.

SEQ ID NO:3 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 35-173 of PilA1.1. The amino acid sequence encodedby SEQ ID NO:3 is SEQ ID NO:4.

SEQ ID NO:7 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 35-173 of PilA1.2. The amino acid sequence encodedby SEQ ID NO:7 is SEQ ID NO:8.

SEQ ID NO:11 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 35-173 of PilA1.3. The amino acid sequence encodedby SEQ ID NO:1 I is SEQ ID NO:12.

SEQ ID NO:15 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 35-171 of PilA1.4. The amino acid sequence encodedby SEQ ID NO:15 is SEQ ID NO:16.

SEQ ID NO:19 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 35-170 of PilA1.5. The amino acid sequence encodedby SEQ ID NO:19 is SEQ ID NO:20.

SEQ ID NO:23 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 34-119 of PilA2.1. The amino acid sequence encodedby SEQ ID NO:23 is SEQ ID NO:24.

SEQ ID NO:27 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 31-116 of PilA2.2. The amino acid sequence encodedby SEQ ID NO:27 is SEQ ID NO:28.

SEQ ID NO:31 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 32-164 of PilA3.1. The amino acid sequence encodedby SEQ ID NO:31 is SEQ ID NO:32.

SEQ ID NO:35 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 32-164 of PilA3.2. The amino acid sequence encodedby SEQ ID NO:35 is SEQ ID NO:36.

SEQ ID NO:39 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 32-162 of PilA3.3. The amino acid sequence encodedby SEQ ID NO:39 is SEQ ID NO:40.

SEQ ID NO:43 is a nucleotide sequence comprising an antigenic fragmentencoding amino acids 36-272 of PilA4.1. The amino acid sequence encodedby SEQ ID NO:43 is SEQ ID NO:44.

In accordance with the invention, such polypeptides and fragments areuseful as immunogens and also as diagnostic tools to aid in thedetection of antibodies that react with type IV pilin of C. difficilefrom a biological sample from a subject suspected of being infected, orat risk for infection, previously infected or immunized with a type IVpilin immunogen.

Methods of Inducing an Immune Response.

The present invention also includes methods of inducing an immuneresponse comprising administering to a subject in need thereof animmunologically-effective amount of a vaccine comprising Clostridiumdifficile type IV pilin or an antigenic fragment or variant thereof.

In certain aspects of the invention, the vaccine is administered alonein a single dose or administered in sequential doses.

In some embodiments, a combination of C. difficile type IV pilins orantigenic fragments or variants thereof is administered, to provideprotection against a broad spectrum of C. difficile strains, orparticular strains that are more prevalent. In some embodiments, thecombination is administered as a single, multivalent vaccinecomposition. In other embodiments, the type IV pilins or antigenicfragments or variants thereof are administered in more than one vaccinecomposition.

In some embodiments, a type IV pilin or antigenic fragment or variantthereof is conjugated, either genetically or chemically to one or moretype IV pilins or antigenic fragments or variants thereof, another C.difficile antigen, a bacterial antigen, toxin or the like, andadministered as a conjugate vaccine.

In some embodiments, one of the following combinations is administered:a combination comprising one or more PilA1, one or more PilA2, one ormore PilA3 and one or more PilA4 proteins, variants or antigenicfragment thereof, a combination comprising one or more PilA1, one ormore PilA2, one or more PilA3 proteins, variants or antigenic fragmentsthereof, a combination comprising one or more PilA1, one or more PilA2,one or more PilA4 proteins, variants or antigenic fragments thereof, acombination comprising one or more PilA1, one or more PilA3, one or morePilA4 proteins, variants or antigenic fragments thereof, a combinationcomprising one or more PilA2, one or more PilA3, one or more PilA4proteins, variants or antigenic fragments thereof, a combinationcomprising one or more PilA1 and one or more PilA2 proteins, variants orantigenic fragments thereof, a combination comprising one or more PilA1and one or more PilA3 proteins, variants or antigenic fragments thereof,a combination comprising one or more PilA1 and one or more PilA4proteins, variants or antigenic fragments thereof, a combinationcomprising one or more PilA2 and one or more PilA3 proteins, variants orantigenic fragments thereof, a combination comprising one or more PilA2and one or more PilA4 proteins, variants or antigenic fragments thereof,and a combination comprising one or more PilA3 and one or more PilA4proteins, variants or antigenic fragments thereof.

In some embodiments, PilA1 comprises a peptide selected from the groupconsisting of a PilA1.1 antigenic fragment (amino acids amino acids35-173 of SEQ ID NO:2), a PilA1.2 antigenic fragment (amino acids aminoacids 35-173 of SEQ ID NO:6), a PilA1.3 antigenic fragment (amino acidsamino acids 35-173 of SEQ ID NO:10), a PilA1.4 antigenic fragment (aminoacids 35-171 of SEQ ID NO:14), a PilA1.5 antigenic fragment (amino acids35-170 of SEQ ID NO:18) and combinations thereof. In some embodiments,PilA1 includes all the PilA1 allele fragments listed above.

In some embodiments, PilA2 comprises a peptide selected from the groupconsisting of a PilA2.1 antigenic fragment (amino acids 34-119 of SEQ IDNO: 22), a PilA2.2 antigenic fragment (amino acids 31-116 of SEQ IDNO:26) and combinations thereof. In some embodiments, PilA2 includes allthe PilA2 allele fragments listed above.

In some embodiments, PilA3 comprises a peptide selected from the groupconsisting of a PilA3.1 antigenic fragment (amino acids 32-164 of SEQ IDNO: 30), a PilA3.2 antigenic fragment (amino acids 32-164 of SEQ IDNO:34), a PilA3.3 antigenic fragment (amino acids 32-162 of SEQ IDNO:38) and combinations thereof. In some embodiments, PilA3 includes allthe PilA3 allele fragments listed above.

In some embodiments, PilA4 comprises a peptide selected from the groupconsisting of a PilA4.1 antigenic fragment (amino acids 36-272 of SEQ IDNO: 42).

In some embodiments, the type IV pilin is from a C. difficile strainselected from the group consisting of C. difficile CD196, C. difficileCP 107932, C. difficile QCD-32g58, C. difficile QCD-37x79, C. difficileQCD-66c26, C. difficile QCD-76w55, C. difficile QCD-97b34, C. difficileR20291, C. difficile QCD-63q42, C. difficile QCD-23m63, C. difficile630, C. difficile ATCC 43255 and combinations thereof.

In some embodiments, the type IV pilin is selected from the groupconsisting of SEQ ID NO:2; SEQ ID NO:6; SEQ ID NO:10; SEQ ID NO:14; SEQID NO:18; SEQ ID NO:22; SEQ ID NO:26; SEQ ID NO:30; SEQ ID NO:34; SEQ IDNO:38; SEQ ID NO:42, variants thereof, antigenic fragments thereof, andcombinations thereof.

In some embodiments, a combination of PilA1 type IV pilin isadministered. In some embodiments, one of the following combinations isadministered: a combination comprising SEQ ID NO:2, variants orantigenic fragments thereof; a combination comprising SEQ ID NO:6,variants or antigenic fragments thereof; a combination comprising SEQ IDNO:10, variants or antigenic fragments thereof; a combination comprisingSEQ ID NO:14, variants or antigenic fragments thereof; and a combinationcomprising SEQ ID NO:18, variants or antigenic fragments thereof.

In some embodiments, a combination of SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:10, SEQ ID NO:14 and SEQ ID NO:18, variants or antigenic fragmentsthereof is administered.

In some embodiments, the combination comprises at least four type IVpilins. In some embodiments, the type IV pilins are PilA1 alleles,variants or antigenic fragments thereof. In some embodiments, one of thefollowing combinations is administered: a combination of SEQ ID NO:2,SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:18 or variants or antigenicfragments thereof; a combination of SEQ ID NO:2, SEQ ID NO:6, SEQ IDNO:10 and SEQ ID NO:14, or variants or antigenic fragments thereof; acombination of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:18or variants or antigenic fragments thereof; a combination of SEQ IDNO:2, SEQ ID NO:6, SEQ ID NO:14 and SEQ ID NO:18 or variants orantigenic fragments thereof; and a combination of SEQ NO:6, SEQ IDNO:10, SEQ ID NO:14 and SEQ ID NO:18 or variants or antigenic fragmentsthereof.

In some embodiments, the combination comprises at least three type IVpilins. In some embodiments, the type IV pilins are PilA1 alleles,variants or antigenic fragments thereof. In some embodiments, one of thefollowing combinations is administered: a combination of SEQ ID NO:2,SEQ ID NO:6 and SEQ ID NO:10, or variants or antigenic fragmentsthereof; a combination of SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:14, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:2,SEQ ID NO:6 and SEQ ID NO:18, or variants or antigenic fragmentsthereof; a combination of SEQ ID NO:2, SEQ ID NO:10 and SEQ ID NO:14, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:2,SEQ ID NO:10 and SEQ ID NO:18, or variants or antigenic fragmentsthereof; a combination of SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:18, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:6,SEQ ID NO:10 and SEQ ID NO:14, or variants or antigenic fragmentsthereof; a combination of SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:18, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:6,SEQ ID NO:14 and SEQ ID NO:18, or variants or antigenic fragmentsthereof; and a combination of SEQ ID NO:10, SEQ ID NO:14 and SEQ IDNO:18, or variants or antigenic fragments thereof.

In some embodiments, the combination comprises at least three type IVpilins that are PilA3 alleles, variants or antigenic fragments thereof.In some embodiments: a combination of SEQ ID NO:30, SEQ ID NO:34 and SEQID NO:38, or variants or antigenic fragments thereof is administered. Insome embodiments, the combination comprises at least two type IV pilins.In some embodiments, the type IV pilins are PilA1 alleles, variants orantigenic fragments thereof. In some embodiments, one of the followingcombinations is administered: a combination of SEQ ID NO:2 and SEQ IDNO:6, or variants or antigenic fragments thereof; a combination of SEQID NO:2 and SEQ ID NO:10, or variants or antigenic fragments thereof; acombination of SEQ ID NO:2 and SEQ NO:14, or variants or antigenicfragments thereof; a combination of SEQ ID NO:2 and SEQ ID NO:18, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:6and SEQ ID NO:10, or variants or antigenic fragments thereof; acombination of SEQ ID NO:6 and SEQ ID NO:14, or variants or antigenicfragments thereof; a combination of SEQ ID NO:6 and SEQ ID NO:18, orvariants or antigenic fragments thereof; a combination of SEQ ID NO:10and SEQ ID NO:14, or variants or antigenic fragments thereof; acombination of SEQ ID NO:10 and SEQ ID NO:18, or variants or antigenicfragments thereof; and a combination of SEQ ID NO:14 and SEQ ID NO:18,or variants or antigenic fragments thereof.

In some embodiments, the combination comprises at least two type IVpilins that are PilA2 alleles, variants or antigenic fragments thereof.In some embodiments: a combination of SEQ ID NO:22 and SEQ ID NO:26 orvariants or antigenic fragments thereof is administered.

In some embodiments, the combination comprises at least two type IVpilins that are PilA3 alleles, variants or antigenic fragments thereof.In some embodiments, one of the following combinations is administered:a combination of SEQ ID NO:30 and SEQ ID NO:34 or variants or antigenicfragments thereof; a combination of SEQ ID NO:30 and SEQ ID NO:38, orvariants or antigenic fragments thereof; and a combination of SEQ IDNO:34 and SEQ ID NO:38 or variants or antigenic fragments thereof.

In some embodiments, a combination comprising a PilA1, PilA2, PilA3 andPilA4 protein, variant or antigenic fragment thereof is administered.

In some embodiments, the antigenic fragment of the PILA1 proteincomprises a peptide selected from the group consisting of amino acids35-173 of SEQ ID NO:2; amino acids 35-173 of SEQ ID NO:6; amino acids35-173 of SEQ ID NO:10; amino acids 35-171 of SEQ ID NO:14; amino acids35-170 of SEQ ID NO:18 and combinations thereof.

In some embodiments, the antigenic fragment of the PILA2 proteincomprises a peptide selected from the group consisting of: amino acids34-119 of SEQ ID NO:22; amino acids 31-116 of SEQ ID NO:26 andcombinations thereof.

In some embodiments, the antigenic fragment of the PILA3 proteincomprises a peptide selected from the group consisting of: amino acids32-164 of SEQ ID NO:30; amino acids 32-164 of SEQ ID NO:34; amino acids32-162 of SEQ ID NO:38; and combinations thereof.

In some embodiments, the antigenic fragment of the PILA4 proteincomprises amino acids 36-272 of SEQ ID NO:42.

In some embodiments, the type IV pilins, variants or antigenic fragmentsthereof for use in the methods of the invention are recombinantlyproduced. In some embodiments, the type IV pilins, variants or antigenicfragments thereof are produced in E. coli using genetically engineerednucleic acids optimized for high level expression using preferred E.coli codons.

As used herein, an immunologically-effective amount is an amountsufficient to induce an immune response in the subject.

As used herein, an “immune response” is the physiological response ofthe subject's immune system to an immunizing composition. An immuneresponse may include an innate immune response, an adaptive immuneresponse, or both. In some embodiments of the present invention, theimmune response is a protective immune response. A protective immuneresponse confers immunological cellular memory upon the subject, withthe effect that a secondary exposure to the same or a similar antigen ischaracterized by one or more of the following characteristics: shorterlag phase than the lag phase resulting from exposure to the selectedantigen in the absence of prior exposure to the immunizing composition;production of antibody which continues for a longer period thanproduction of antibody resulting from exposure to the selected antigenin the absence of prior exposure to the immunizing composition; a changein the type and quality of antibody produced in comparison to the typeand quality of antibody produced upon exposure to the selected antigenin the absence of prior exposure to the immunizing composition; a shiftin class response, with IgG antibodies appearing in higherconcentrations and with greater persistence than IgM, than occurs inresponse to exposure to the selected antigen in the absence of priorexposure to the immunizing composition; an increased average affinity(binding constant) of the antibodies for the antigen in comparison withthe average affinity of antibodies for the antigen resulting fromexposure to the selected antigen in the absence of prior exposure to theimmunizing composition; and/or other characteristics known in the art tocharacterize a secondary immune response.

In some embodiments, the vaccines of the invention are administered witha pharmaceutically acceptable carrier, such that it provides hostimmunity against an infection.

The term “carrier” denotes an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitatethe application of the vaccine. The characteristics of the carrierdepend on the nature of the vaccine and the route of administration.Physiologically and pharmaceutically-acceptable carriers includediluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials. The term “pharmaceutically acceptable” is used to refer to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism.

In practicing immunization protocols for treatment and/or prevention, animmunologically-effective amount of type IV pilin or a variant orantigenic fragment thereof is administered to a subject. The particulardosage depends upon the age, weight, sex and medical condition of thesubject o be treated, as well as on the method of administration.

The vaccines of the invention can be administered by either single ormultiple dosages of an effective amount. In some embodiments, aneffective amount of the type IV pilin of the invention can vary from0.01-5,000 μg/ml per dose. In other embodiments, an effective amount ofthe type IV pilin can vary from 0.1-500 μg/ml per dose, and in otherembodiments, it can vary from 10-300 μg/ml per dose. In one embodiment,the dosage of type IV pilin will range from about 10 μg to about 1000μg. In another embodiment, the amount administered will be between about20 μg and about 500 μg. In some embodiments, the amount administeredwill be between about 75 μg and 250 μg. Greater doses may beadministered on the basis of body weight. The exact dosage can bedetermined by routine dose/response protocols known to one of ordinaryskill in the art.

In some embodiments, the amount of the type IV pilin that provides animmunologically-effective amount for vaccination against infection isfrom about 1 μg or less to about 5000 μg or more. In some embodiments,it is from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45or 50 μg is to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 μg per kgbody weight. In one embodiment, the immunologically-effective amount forvaccination against bacterial infection is from 10 μg to 1000 μg.

The term “subject” as used herein, refers to animals, such as mammals.For example, mammals contemplated include humans, primates, dogs, cats,sheep, cattle, goats, pigs, horses, chickens, mice, rats, rabbits,guinea pigs, and the like. The teems “subject”, “patient”, and “host areused interchangeably.

In some embodiments, the subject is a human. In some embodiments, thesubjects are patients who are at high risk of C. difficile infections.In some embodiments, the subjects are selected from the group consistingof elderly patients in healthcare or nursing care settings, patients whohave undergone antibiotic treatment of unrelated infections, arecurrently undergoing antibiotic treatment or are about to undergoantibiotic treatment, patients in healthcare settings, patients who havepreviously been infected with C. difficile or who have experienced CDADsymptoms. In some embodiments, the subjects are outpatients. In someembodiments, the subjects are healthy individuals. In some embodiments,the subjects are at risk of C. difficile infection because of theirclose contact with an infected individual or exposure to surroundingsthat might be infected with C. difficile or infection causing sporesthereof.

In some embodiments, the subjects include patients that have receivedbroad spectrum antibiotics, such as hospitalized elderly patients,nursing home residents, chronically ill patients, cancer patients, AIDSpatients, patients in intensive care units, and patients receivingdialysis treatment.

The vaccine of the present invention may confer resistance toClostridium difficile by either passive immunization or activeimmunization. In one embodiment of passive immunization, the vaccine isprovided to a subject (i.e. a human or mammal), and the elicitedantisera is recovered and directly provided to a recipient suspected ofhaving an infection caused by C. difficile.

In some embodiments of passive immunization, the C. difficile immuneglobulin is administered in amounts ranging from 100 μg/kg-100 mg/kg, or1-50 mg/kg, for example, about 15 mg/kg, depending on donor titer. Theimmune globulin can be administered in, e.g., one or two doses. aninitial dose can be administered for treatment and a second dose can beadministered to prevent relapse.

The administration of the vaccine (or the antisera which it elicits) maybe for either a “prophylactic” or “therapeutic” purpose. When providedprophylactically, the vaccine is provided in advance of any symptom ofC. difficile infection. The prophylactic administration of the vaccineserves to prevent or attenuate any subsequent infection. When providedtherapeutically, the vaccine is provided upon the detection of a symptomof actual infection. The therapeutic administration of the vaccineserves to attenuate any actual infection. In some embodiments,administration of the vaccine of the invention attenuates C. difficilecolonization and disease in the subject. In some embodiments,administration of the vaccine of the invention prevents C. difficilecolonization and disease in the subject.

The vaccines (or antisera which it elicits) can be provided either priorto the onset of infection (so as to prevent or attenuate an anticipatedinfection) or after the initiation of an actual infection.

In some embodiments, the vaccines are administered with other vaccinestargeting other components of C. difficile. In some embodiments, thevaccines are administered in conjunction with vaccines comprising toxoidpreparations of enterotoxins A (TcdA) and B (Tea) of C. difficile.

In some embodiments, the subject is co-administered with agents used totreat C. difficile infection, such as metronidazole and/or vancomycin inconjunction with methods as described herein.

The invention also provides a method for inducing an immune responsewhich comprises administering to a subject, suspected of being at riskfor infection caused by C. difficile, an immunologically-effectiveamount of an antisera elicited from the exposure of a second individualto a vaccine of the invention, such that it provides host immunity tothe infection.

The vaccine of the invention can be administered to mammals of any age.In some embodiments, the vaccines can be administered as a single doseor in a series including one or more boosters. In some embodiments, thetime interval between the first and second vaccinations is one week, twoweeks, three weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, one year,1.5 years and two years. In some embodiments, two sequential boosterimmunizations are administered.

In some embodiments, the immunization schedule would involve a primaryseries of three immunizations with a spacing of 1-2 months between thedoses. In some embodiments, a booster dose could be administered ˜6-12months later.

Conjugate Vaccines

In one aspect, the present invention is further directed to a conjugatevaccine comprising a C. difficile type IV pilin, an antigenic fragmentor a variant thereof.

The conjugation can be either through chemical or genetic means. Thegenetic or chemical conjugation encompasses coupling the type IV pilineither through gene fusion or chemically to another entity, for example,using cross-linkers, to increase the immune response. Standardtechniques and methods can be employed to make the conjugate vaccines ofthe invention.

In some embodiments, the C. difficile type IV pilin, antigenic fragmentor variant thereof is conjugated to another C. difficile type W pilin,antigenic fragment or variant thereof. In some embodiments, conjugationis to a bacterial toxin. In some embodiments, it is conjugated to anontoxic variant of a bacterial toxin. In some embodiments, it isconjugated to a nontoxic variant of enterotoxin A (TcdA) or B (TcdB).Other toxins include tetanospasmin, alpha toxin, enterotoxin, botoxdiphtheria toxin, anthrax toxin, listeriolysin O, streptolysin,leukocidin (Panton-Valentine leukocidin), Staphylococcus aureusalpha/beta/delta, exfoliatin, toxic shock syndrome toxin, SEB), cordfactor, diphtheria toxin, shiga toxin, verotoxin/shiga-like toxin (E.coli), E. coli heat-stable enterotoxin/enterotoxin, cholera toxin,pertussis toxin, Pseudomonas exotoxinextracellular adenylate cyclase,type I (Superantigen), type II (Pore forming toxins), type III (ABtoxin/AB5), lipopolysaccharide (Lipid A), Bacillus thuringiensis deltaendotoxin, clumping factor A, and fibronectin binding protein A.

In some embodiments, the C. difficile type IV pilin, antigenic fragmentor variant thereof conjugated to a bacterial toxin is selected from thegroup consisting of PilA1 , PilA2, PilA3 and PilA4. In some embodiments,the C. difficile type IV pilin, antigenic fragment or variant thereofconjugated to a bacterial toxin is PilA2.

In some embodiments, a C. difficile type IV pilin, antigenic fragment orvariant thereof is conjugated to a polysaccharide, using techniquesknown in the art.

A multivalent vaccine may also be prepared by mixing the C. difficiletype IV pilin conjugate with other antigens, including other C.difficile type IV pilins and conjugates thereof, other C. difficileantigens and conjugates thereof, antigens against other organisms andconjugates thereof, bacterial toxins as discussed above and conjugatesthereof, and/or other polysaccharides and conjugates thereof, usingtechniques known in the art. In some embodiments, the invention isdirected to a multivalent vaccine comprising a mixture of C. difficiletype IV pilin conjugates derived from various C. difficile strains, eachconjugate comprising a type IV pilin characteristic of the strain.

Methods for making conjugate vaccines are described in, for example, USPatent Application Publication No, 20090028889. Techniques to conjugatea type IV pilin include, in part, coupling through available functionalgroups (such as amino, carboxyl, thio and aldehyde groups). See, e.g.,Hermanson, Bioconjugate Techniques (Academic Press; 1992); Aslam andDent, eds. Bioconjugation: Protein coupling Techniques for theBiomedical Sciences (MacMillan: 1998); S. S. Wong, Chemistry of ProteinConjugate and Crosslinking CRC Press (1991), and Brenkeley et al., BriefSurvey of Methods for Preparing Protein Conjugates With Dyes, Haptensand Cross-Linking Agents, Bioconjugate Chemistry 3 #1 (January 1992);Jacob, C. O, et al., Eur. J. Immunol. 16:1057-1062 (1986); Parker, J. M.R. et al., In: Modern Approaches to Vaccines, Chanock, R. M. et al.,eds, pp. 133-138, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1983); Zurawski, V. R, et al., J. Immunol. 121:122-129 (1978);Klipstein, F. A, et al., Infect. Immun. 37:550-557 (1982); Bessler, W.G, Immunobiol. 170:239-244 (1985); Posnett, D. N, et al., J. Biol. Chem.263:1719-1725 (1988); Ghose, A. C, et al., Molec. Immunol. 25:223-230(1988); all of which disclosures are incorporated herein by reference).An example of a conjugate vaccine was developed against Haemophilusinfluenzae (Anderson, P, Infec. and Immunology 39:223-238 (1983); Chu,C, et al., Infect. Immun. 40:245-256 (1983); Lepow, M, Pediat. Infect.Dis. J. 6:804-807 (1987), which disclosures are incorporated herein byreference), Additional methods for producing such a conjugate vaccineare disclosed by European Patent Publication 245,045; U.S. Pat. Nos.4,673,574 and 4,761,283; U.S. Pat. No. 4,789,735; European PatentPublication No. 206,852; U.S. Pat. No. 4,619,828; U.S. Pat. No.4,284,537; U.S. Pat. No. 5,192,540; U.S. Pat. No. 5,370,872; U.S. Pat.No. 5,302,386; and U.S. Pat. No. 5,576,002 all of which disclosures areincorporated herein by reference.

In some embodiments, the conjugate vaccine comprises a type IV pilin,antigenic fragment or a variant thereof selected from the groupconsisting of PilA1, PilA2, PilA3 and PilA4.

In some embodiments, the conjugate vaccine comprises a combination oftype IV pilins, antigenic fragments or variants thereof as describedherein.

In some embodiments, the conjugate vaccine comprises multiple alleles ofa type IV pilin, antigenic fragment or variant thereof in combinationsas described herein. For example, in some embodiments, multiple allelesare genetically conjugated to each other to make a fusion protein. Insome embodiments, the conjugate vaccine comprises PilA1.1, PilA1.2,PilA1.3, PilA1.4 and PilA1.5 or antigenic fragments or variants thereof.In some embodiments, the conjugate vaccine comprises PilA2.1 and PilA2.2or antigenic fragments or variants thereof. In some embodiments, theconjugate vaccine comprises PilA3.1, PilA3.2 and PilA3.3 or antigenicfragments or variants thereof.

Vaccine Compositions

As would be understood by one of ordinary skill in the art, when the C.difficile type IV pilin of the present invention is provided to asubject, it may be in a composition which may contain salts, buffers,adjuvants, or other substances which are desirable for improving theefficacy of the composition. Adjuvants are substances that can be usedto specifically augment a specific immune response. Normally, theadjuvant and the composition are mixed prior to presentation to theimmune system, or presented separately, but into the same site of theanimal being immunized. Adjuvants can be loosely divided into severalgroups based upon their composition. These groups include oil adjuvants(for example, Freund's complete and incomplete), mineral salts (forexample, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄ (SO₄), silica, kaolin, andcarbon), polynucleotides (for example, poly IC and poly AU acids), andcertain natural substances (for example, wax D from Mycobacteriumtuberculosis, as well as substances found in Corynebacterium parvum, orBordetella pertussis, and members of the genus Brucella. Adjuvants aredescribed by Warren et al. (Ann. Rev. Biochem., 4:369-388, 1986), theentire disclosure of which is hereby incorporated by reference.

In some embodiments of the invention, conventional adjuvants can beadministered together with the type IV pilin. In some embodiments, theadjuvants are saponins such as, for example, Quil A. (Superfos A/S,Denmark). In some embodiments, monophosphoryl lipid A plus trehalosedimycolate (Ribi-700; Ribi Immunochemical Research, Hamilton, Mont.) isused as an adjuvant.

The vaccines can be formulated into liquid preparations for, e.g.,nasal, rectal, buccal, vaginal, peroral, intragastric, mucosal,perlinqual, alveolar, gingival, olfactory, or respiratory mucosaadministration. Suitable font's for such administration includesolutions, suspensions, emulsions, syrups, and elixirs. The vaccines canalso be formulated for parenteral, subcutaneous, intradermal,intramuscular, intraperitoneal or intravenous administration, injectableadministration, sustained release from implants, or administration byeye drops. Suitable forms for such administration include sterilesuspensions and emulsions. Such vaccines can be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, and the like. The vaccines can also belyophilized. The vaccines can contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Texts, such as Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 20th edition (Jun. 1, 2003) andRemington's Pharmaceutical Sciences, Mack Pub. Co.; 18^(th) and 19^(th)editions (December 1985, and June 1990, respectively), incorporatedherein by reference in their entirety, can be consulted to preparesuitable preparations. Such preparations can include complexing agents,metal ions, polymeric compounds such as polyacetic acid, polyglycolicacid, hydrogels, dextran, and the like, liposomes, microemulsions,micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

In some embodiments, the vaccine of the invention is administeredparenterally. Parenteral vehicles include phosphate buffered saline,sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. In some embodiments, thevaccines for parenteral administration may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers or occlusive dressings can be used to increaseskin permeability and enhance antigen absorption. Suspensions may beformulated according to methods well known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a parenterally acceptable diluent or solvent, such as asolution in 1,3-butanediol. Suitable diluents include, for example,water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed conventionally as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectable preparations.

Liquid dosage fonts for oral administration may generally comprise aliposome solution containing the liquid dosage form. Suitable forms forsuspending liposomes include emulsions, suspensions, solutions, syrups,and elixirs containing inert diluents commonly used in the art, such aspurified water. Besides the inert diluents, such compositions can alsoinclude adjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

In some embodiments, the vaccines are provided as liquid suspensions oras freeze-dried products. Suitable liquid preparations include, e.g.,isotonic aqueous solutions, suspensions, emulsions, or viscouscompositions that are buffered to a selected pH. Transdermalpreparations include lotions, gels, sprays, ointments or other suitabletechniques. If nasal or respiratory (mucosal) administration is desired(e.g., aerosol inhalation or insufflation), compositions can be in aform and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or adose having a particular particle size, as discussed below.

When in the form of solutions, suspensions and gels, in someembodiments, the formulations contain a major amount of water(preferably purified water) in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers,dispersing agents, buffering agents, preservatives, wetting agents,jelling agents, colors, and the like can also be present.

In some embodiments, the compositions are isotonic with the blood orother body fluid of the recipient. In some embodiments, the isotonicityof the compositions can be attained using sodium tartrate, propyleneglycol or other inorganic or organic solutes. In some embodiments,sodium chloride is used. In some embodiments, buffering agents can beemployed, such as acetic acid and salts, citric acid and salts, boricacid and salts, and phosphoric acid and salts. In some embodiments ofthe invention, phosphate buffered saline is used for suspension.

In some embodiments, the viscosity of the compositions can be maintainedat the selected level using a pharmaceutically acceptable thickeningagent. In some embodiments, methylcellulose is used because it isreadily and economically available and is easy to work with. Othersuitable thickening agents include, for example, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and thelike. The concentration of the thickener can depend upon the agentselected. In some embodiments, viscous compositions are prepared fromsolutions by the addition of such thickening agents.

In some embodiments, a pharmaceutically acceptable preservative can beemployed to increase the shelf life of the compositions. Benzyl alcoholcan be suitable, although a variety of preservatives including, forexample, parabens, thimerosal, chlorobutanol, or benzalkonium chloridecan also be employed. A suitable concentration of the preservative canbe from 0.02% to 2% based on the total weight although there can beappreciable variation depending upon the agent selected.

In some embodiments, pulmonary delivery of the vaccine can also beemployed. In some embodiments, the vaccine is delivered to the lungs ofa mammal while inhaling and traverses across the lung epithelial liningto the blood stream. A wide range of mechanical devices designed forpulmonary delivery of therapeutic products can be employed, includingbut not limited to nebulizers, metered dose inhalers, and powderinhalers, all of which are familiar to those skilled in the art. Thesedevices employ formulations suitable for the dispensing of theconjugate. Typically, each formulation is specific to the type of deviceemployed and can involve the use of an appropriate propellant material,in addition to diluents, adjuvants and/or carriers useful in therapy.

In embodiments where the vaccine is prepared for pulmonary delivery inparticulate form, it has an average particle size of from 0.1 μM or lessto 10 μm or more. In some embodiments, it has an average particle sizeof from about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm to about 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, or 9.5 μm for pulmonary delivery. Pharmaceutically acceptablecarriers for pulmonary delivery of the vaccine include carbohydratessuch as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol.Other ingredients for use in formulations can include DPPC, DOPE, DSPCand DOPC. Natural or synthetic surfactants can be used, includingpolyethylene glycol and dextrans, such as cyclodextran and other relatedenhancers, as well as cellulose and cellulose derivatives, and aminoacids can also be used. Liposomes, microcapsules, microspheres,inclusion complexes, and other types of carriers can also be employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, typically comprise the peptide dissolved or suspended inwater at a concentration of about 0.01 or less to 100 mg or more ofpeptide per mL of solution, preferably from about 0.1, 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, or 90 mg of peptide per mL of solution. The formulationcan also include a buffer and a simple sugar (e.g., for proteinstabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the conjugate caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such chlorofluorocarbon, ahydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons, such astrichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, andcombinations thereof. Suitable surfactants include sorbitan trioleate,soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing the peptide, optionallyincluding a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in an amount that facilitates dispersal of thepowder from the device, typically from about 1 wt. % or less to 99 wt. %or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30,35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. %of the formulation.

Diagnostic Methods

In some embodiments, the invention relates to type IV pilinpolynucleotides, proteins or antibodies reactive specifically againstthe type IV pilins for use as diagnostic reagents. Detection of DNA,mRNA (or cDNA), protein levels, or antibodies directed against type IVpilins will provide a diagnostic tool that can add to or define adiagnosis of infection or susceptibility to infection. In someembodiments, the detection of one or more type IV pilins will direct themedical practitioner to set an appropriate course of treatment for thepatient.

Nucleic acids for diagnosis may be obtained, for example, from asubject's bodily sample, such as, for example, the stool. Nucleic acidlevels (DNA, mRNA, cDNA) can be assayed by comparison to a standard orcontrol level from an uninfected and/or infected individual. The nucleicacids can be used directly for detection or can amplified enzymaticallyby using PCR or other amplification techniques prior to analysis.

In some embodiments, expression is measured at the RNA level using anyof the methods well known in the art for the quantitation ofpolynucleotides; for example, RT-PCR, RNase protection, Northernblotting, array analysis, and other hybridization methods may beutilized. Assay techniques that may be used to determine the level of atype IV pilin protein in a sample derived from a host includeradioimmunoassays, competitive-binding assays, Western blot analysis andELISA assays.

In some embodiments, the invention is directed to methods for detectingC. difficile infection in a subject. In some embodiments, the methodcomprises obtaining a bodily sample from the subject; and analyzing thesample for the presence of a type IV pilin from C. difficile or anantibody that binds thereto. In some embodiments, the sample is a stoolsample, a blood sample, a serum sample, a urine sample, and a salivasample.

In some embodiments, the type IV pilin nucleic acid is detected. In someembodiments, type IV pilin protein is detected. In some embodiments, anantibody binding to type IV pilin is detected.

Other C. difficile markers, such as enterotoxins A (TcdA) and B (TcdB),can also be simultaneously assayed, in accordance with the methods ofthe present invention.

Type IV Pilin Antibodies

The type IV pilins of the invention or their antigenic fragments orvariants thereof, or cells expressing them may also be used asimmunogens to produce antibodies immunospecific for the type IV pilin.

Antibodies generated against the type IV pilin polypeptides can beobtained by administering the polypeptides or antigenic fragments,variants or cells to an animal using routine protocols. In someembodiments, antisera against type IV pilin are generated in a subjectfor use in passive immunotherapy. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique(Kozbor, et al., Immunology Today 4:72 (1983)) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., (1985)).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) may also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies. In some embodiments, the antibodies are humanized.

In some embodiments, the above-described antibodies or antisera isemployed to detect a type IV pilin, for use in passive immunotherapy, orto purify a type IV pilin by affinity chromatography.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not to be construed as a limitation thereof.

EXAMPLE 1

Expression and purification of the type IV pilins from C. difficile. Weused the following novel strategy to express and purify high levels ofrecombinant C. difficile type IV pilins. This method involvesidentification of insoluble and soluble domains of the proteins,engineering of codon optimized synthetic pilin genes which do not existin nature but which result in expression of pilin domains identical tothose found in nature, optimal expression of pilin proteins, andpurification to homogeneity. This method has been successful using twodifferent pilin gene sequences.

We used our understanding of type IV pilin protein structure to identifythe pre-pilin peptidase cleavage sites and hydrophobic matureamino-termini of each pilin gene. For the pilA2 gene found in C.difficile strain 630 and 10 other sequenced strains, we omitted from therecombinant construct the codons for the signal sequence and the first27 residues of the mature protein (FIG. 1). In type IV pilus biogenesisthe former domain is removed prior to pilus assembly and the latterdomain is highly hydrophobic, buried in the core of the pilus, andinterferes with pilin solubility and purification. Antibodies directedagainst this domain are not protective against infection since they donot bind to epitopes expressed on the pilus surface.

Next, using oligonucleotide assembly polymerase chain reaction, wesynthesized the remaining part of the pilA2 gene using codons optimizedfor E. coli to avoid translation limitations imposed by the differencesin codon preference between these unrelated species. This procedureresulted in alteration of 73% of the codons without changing any of thenatural amino acid sequence. This sequence is entirely novel and doesnot occur in nature. We then cloned the truncated optimized pilA2 geneinto the pET30 T7 expression plasmid (Novagen) in frame with ahexahistidine tag (facilitating purification) and an enterokinasecleavage site (for potential removal of the tag, if required). Aftertransformation and induction in E. coli strain BL21, we achievedextraordinarily robust levels of expression of a protein with theexpected molecular mass of 13.8 kDa (FIG. 2). We were able to obtainmore than 70 mg of pure protein from two liters of culture afterone-step affinity purification on a nickel-nitrilotriacetic acid column.

Similarly, we engineered, synthesized and subcloned a recombinant pilA1expression plasmid using the sequence from C. difficile strain 630 as aguide and following the identical strategy of domain omission, codonoptimization, and vector and strain selection used for pilA2. We weresimilarly successful in high yield, high purity expression ofrecombinant PilA1 protein (FIG. 2). In the sequence listing we presentthe sequence of novel recombinant vectors for expression of each variantof PilA1, PilA2, PilA3, and PilA4. In some embodiments, the inventionrelates to identifying, engineering and producing recombinant syntheticC. difficile pilin genes of any type for pilin protein purification anduse in vaccines and for diagnosis.

EXAMPLE 2

Generation of polyclonal antisera against recombinant C. difficilePilA1. We immunized 5 mice each with purified PilA1 and PilA2 pilinproteins using a standard regimen consisting of primary subcutaneousinjection with Freund's complete adjuvant followed by two boosterimmunizations with incomplete adjuvant. Using the purified pilinproteins as antigens, we were able to demonstrate in 4 of 5 mice asignificant immune response against PilA1 (FIG. 3). However, PilA2 wasnot immunogenic using this protocol.

EXAMPLE 3

The efficacy of passive immunization with antibodies to PilA1 inprevention of C. difficile colonization and disease in a murine model.We will determine the efficacy of passive immunization using a recentlyreported murine model of experimental C. difficile colitis. Chen et al.Gastroenterol. 135(6):1984-92 (2008). Female nine-week-old C57BL6 micewill be treated for 3 days with an antibiotic cocktail of kanamycin (0.4mg/ml), gentamicin (0.035 mg/ml), colistin (850 U/ml), metronidazole(0.215 mg/ml), and vancomycin (0.045 mg/ml), administered in thedrinking water. After this treatment, mice will be switched over toregular drinking water for another 3 days. Finally, on day 7, a singledose of clindamycin (10 mg/kg) will be administered by theintraperitoneal route 24 hrs prior to orogastric challenge with C.difficile strain 630. One group of 14 mice will receive twice the LD₅₀(1×10⁴ CFU) of C. difficile strain 630 pre-mixed with normal mouse seraand the other will receive the same dose pre-mixed with pooled immunesera from the four mice with titers against PilA 1 greater than1:10,000. Mice will be weighed daily, scored for the development ofdiarrhea, and followed until they recover, die or require euthanasia.CDAD will be defined as any of the following: diarrhea, loss of 5% ofpre-challenge body weight, or death. The experiment will be repeatedonce. With 28 mice in each group there is an 80% chance of detecting areduction in CDAD, the primary endpoint, from 60% to 20% with a Pvalue<0.05. In addition to monitoring CDAD, we will measure the effectof passive immunization on colonization and excretion of the organism,which will be highly relevant to control of C. difficile in health caresettings.

EXAMPLE 4

The efficacy of immunization with purified pilin in prevention of C.difficile colonization and disease in a murine model. We will establishthe immunogenicity and protective efficacy of immunization with PilA1and PilA2 in the murine model. Groups of six mice each will receivesubcutaneous injections of PBS, PilA1 or PilA2. Fourteen days aftercompletion of the immunization schedule, mice will be treated withantimicrobials and challenged with twice the LD₅₀ (1×10⁴ CFU) of C.difficile strain 630 as described in section above. Mice will be weigheddaily, scored for the development of diarrhea, and followed until theyrecover, die or require euthanasia. CDAD will be defined as any of thefollowing: diarrhea, loss of 5% of pre-challenge body weight, or death.The experiment will be repeated three times for a total of 24 mice ineach group. In addition to monitoring CDAD, we will measure the effectof passive immunization on colonization and excretion of the organism.

1. A method of inducing an immune response, comprising administering toa subject in need thereof an immunologically-effective amount of avaccine comprising a Clostridium difficile type IV pilin or an antigenicfragment or variant thereof, wherein the variant is recognized by anantibody that binds the C. difficile type IV pilin.
 2. The method ofclaim 1, wherein a combination of C. difficile type IV pilins orantigenic fragments or variants thereof are administered.
 3. The methodof claim 1, wherein an antigenic fragment is administered from a type IVpilin selected from the group consisting of: a. SEQ ID NO:2; b. SEQ IDNO:6; c. SEQ ID NO:10; d. SEQ ID NO:14; e. SEQ ID NO:18; f. SEQ IDNO:22; g. SEQ ID NO:26; h. SEQ ID NO:30; i. SEQ ID NO:34; j. SEQ IDNO:38; and k. SEQ ID NO:42.
 4. The method of claim 1, wherein theantigenic fragment comprises a peptide selected from the groupconsisting of: a. amino acids 35-173 of SEQ ID NO:2; b. amino acids35-173 of SEQ ID NO:6; c. amino acids 35-173 of SEQ ID NO:10; d. aminoacids 35-171 of SEQ ID NO:14; e. amino acids 35-170 of SEQ ID NO:18; f.amino acids 34-119 of SEQ ID NO:22; g. amino acids 31-116 of SEQ IDNO:26; h. amino acids 32-164 of SEQ ID NO:30; i. amino acids 32-164 ofSEQ ID NO:34; j. amino acids 32-162 of SEQ ID NO:38; and k. amino acids36-272 of SEQ ID NO:42.
 5. The method of claim 4, wherein the antigenicfragment is recombinantly produced.
 6. The method of claim 5, whereinthe antigenic fragment is produced in E. coli.
 7. The method of claim 6,wherein the antigenic fragment is encoded by a nucleic acid sequenceoptimized to increase expression in E. coli comprising an affinity tagsequence and enzymatic cleavage sequence to facilitate purification. 8.The method of claim 7, wherein the nucleic acid sequence is selectedfrom the group consisting of: a. SEQ ID NO:3; b. SEQ ID NO:7; c. SEQ IDNO:11; d. SEQ ID NO:15; e. SEQ ID NO:19; f. SEQ ID NO:23; g. SEQ IDNO:27; h. SEQ ID NO:31; i. SEQ ID NO:35; j. SEQ ID NO:39; and k. SEQ IDNO:43.
 9. The method of claim 2, wherein the combination comprises anantigenic fragment of SEQ ID NO:2.
 10. The method of claim 9, whereinthe combination further comprises an antigenic fragment of a type IVpilin selected from the group consisting of: a. SEQ ID NO:6; b. SEQ IDNO:10; c. SEQ ID NO:14; d. SEQ ID NO:18; and e. combinations thereof,11. A method for detecting a C. difficile infection in a subject,comprising: obtaining a bodily sample from the subject and analyzing thesample for the presence of a type IV pilin from C. difficile or anantibody that binds thereto.
 12. The method of claim 11, wherein theantibody recognizes an antigenic fragment selected from the groupconsisting of: a. amino acids 35-173 of SEQ ID NO:2; b. amino acids35-173 of SEQ ID NO:6; c. amino acids 35-173 of SEQ ID NO:10; d. aminoacids 35-171 of SEQ ID NO:14; e. amino acids 35-170 of SEQ ID NO:18; f.amino acids 34-119 of SEQ ID NO:22; g. amino acids 31-116 of SEQ IDNO:26; h. amino acids 32-164 of SEQ ID NO:30; i. amino acids 32-164 ofSEQ ID NO:34; j. amino acids 32-162 of SEQ ID NO:38; and k. amino acids36-272 of SEQ ID NO:42.
 13. A vaccine comprising a C. difficile type IVpilin or an antigenic fragment or variant thereof, wherein the variantis recognized by an antibody that binds the C. difficile type IV pilin.14. The vaccine of claim 13, comprising a combination of C. difficiletype IV pilins or antigenic fragments or variants thereof.
 15. Thevaccine of claim 14, wherein said type IV pilins or antigenic fragmentsor variants thereof are conjugated.
 16. The vaccine of claim 14, whereinsaid type IV pilins or antigenic fragments or variants thereof areconjugated genetically.
 17. The vaccine of claim 14, comprising a typeIV pilin selected from the group consisting of: a. SEQ ID NO:2; b. SEQID NO:6; c. SEQ ID NO:10; d. SEQ ID NO:14; e. SEQ ID NO:18; f. SEQ IDNO:22; g. SEQ ID NO:26; h. SEQ ID NO:30; i. SEQ ID NO:34; j. SEQ IDNO:38; k. SEQ ID NO:42; and l. combinations thereof.
 18. The vaccine ofclaim 14, comprising a C. difficile type IV pilin antigenic fragment.19. The vaccine of claim 18, wherein the antigenic fragment comprises apeptide selected from the group consisting of: a. amino acids 35-173 ofSEQ ID NO:2; b. amino acids 35-173 of SEQ ID NO:6; c. amino acids 35-173of SEQ ID NO:10; d. amino acids 35-171 of SEQ ID NO:14; e. amino acids35-170 of SEQ ID NO:18; f. amino acids 34-119 of SEQ ID NO:22; g. aminoacids 31-116 of SEQ ID NO:26; h. amino acids 32-164 of SEQ ID NO:30; i.amino acids 32-164 of SEQ ID NO:34; j. amino acids 32-162 of SEQ IDNO:38; k. amino acids 36-272 of SEQ ID NO:42; and l. combinationsthereof.
 20. The method vaccine of claim 19, wherein the antigenicfragment is recombinantly produced. 21-25. (canceled)